The human LT system. VII. Release of soluble forms and delivery to target L-929 cells by lectin-preactivated human lymphocytes in the absence of protein synthesis and secretory processes.

We have investigated the effects of inhibitors of cellular protein synthesis (emetine, cycloheximide) and secretion (colchicine, cytochalasin B) on the capacity of primary or secondary lectin-activated human lymphocytes to release LT molecules or to cause lectin-induced destruction (LICC) of murine L-929 cells in vitro. Our findings reveal: (a) agents which inhibit protein synthesis or secretion block the release of LT activity into the supernatant and LICC when primary lectin-stimulated human adenoid lymphocytes are employed as effector cells; (b) these same agents are ineffective at blocking LT release or LICC when 3- or 5-day lectin-prestimulated lymphocytes are employed; and (c) anti-human α-LT serum blocks LICC of L-929 cells mediated by primary or secondary lectin-activated human lymphocytes. The difference in participation of effector cellular processes in LICC between primary and secondary lectin-stimulated cells correlates with the findings that preactivated lymphoid cells possess high levels of preformed intracellular, as well as membrane associated, LT molecules, and that release of these materials into the supernatant or delivery to the target cell can occur independently of active protein biosynthesis or classical secretory systems.     

Similar molecular requirements for antigen receptor-triggered secretion of interferon and granule enzymes by cytolytic T lymphocytes

At least two biologically significant responses are triggered by the crosslinking of the T-cell receptor (TcR) on the surface of cloned cytotoxic T lymphocytes (CTL): synthesis and secretion of macrophage-activating factor(s) (MAF) that can be attributed to interferon-gamma (IFN) and release of preformed cytolytic granules. We directly compared the molecular requirements for synthesis and secretion of IFN and secretion of granule enzymes triggered in the same cell by the same activating ligand (antigen or monoclonal antibody (mAb) to TcR). An increase in the surface density of activating ligand (immobilized anti-TcR mAb) enhanced both secretion of IFN and secretion of granules. Secretion of IFN occurred immediately after synthesis: only low (but detectable) levels of IFN were detected in cell cytosolic or particulate fractions isolated from Percoll gradients of lysed CTL, while very high levels of IFN were found in the stimulated CTL culture fluids. Inhibitors of RNA synthesis and protein synthesis blocked secretion of IFN, but did not inhibit release of preformed cytolytic granules. The requirement for TcR crosslinking in triggering both secretion of granules and secretion of IFN from CTL was pharmacologically reproduced by the synergistic action of PMA, a protein kinase C activator, and the Ca2+ ionophore A23187. Both secretion of IFN and secretion of granules were absolutely dependent upon extracellular Ca2+: EGTA completely blocked both TcR- and PMA/A23187-induced secretion of IFN and exocytosis of granules. These studies suggest that similar molecular mechanisms are involved in secretion of newly synthesized IFN and secretion of preformed cytolytic granules. One notable difference between the molecular requirements for the two secretory events was a much lower concentration requirement for PMA for IFN synthesis and secretion than for granule secretion in the synergistic interactions with A23187. Implications of these studies for the exocytosis model of cell-mediated cytotoxicity are discussed.     

Induction of macrophage lysosomal hydrolase synthesis and secretion by beta-1,3-glucan

Studies were undertaken to elucidate the active component in zymosan necessary to induce the delayed-onset synthesis and secretion of representative lysosomal hydrolases, hexosaminidase, and beta-glucuronidase in macrophages. Resident mouse peritoneal macrophages were challenged with zymosan particles and particulate beta-1,3-glucan, the major subcomponent of zymosan. Zymosan was found to induce a rapid secretion of preformed hexosaminidase with maximal release (75%) occurring 6 hr after the addition of zymosan. By contrast, beta-1,3-glucan was totally inactive in this respect. However, both zymosan and beta-1,3-glucan were found to induce the delayed-onset synthesis and secretion of hexosaminidase and beta-glucuronidase while maintaining constant cellular enzyme levels over a 5-day period following the addition of stimulus. These late responses were almost totally blocked by a noncytolytic concentration of cycloheximide, indicating their dependence on de novo protein synthesis. Mannan, the second major subcomponent of zymosan, had no effect on either immediate secretion or delayed-onset synthesis and secretion of hexosaminidase. These results suggest that the induction of the delayed-onset synthesis and secretion of the lysosomal hydrolases by zymosan may be dependent on the glucan subcomponent of zymosan. Moreover, it would also appear that the release of preformed lysosomal enzymes is not the trigger for the delayed-onset synthesis and secretion of hexosaminidase.     

The Adaptor 3BP2 Is Required for Early and Late Events in Fc{varepsilon}RI Signaling in Human Mast Cells

Adaptor molecules are essential in organizing signaling molecules and in coordinating and compartmentalizing their activity. SH3-binding protein 2 (3BP2) is a cytoplasmic adaptor protein mainly expressed by hematopoietic cells that has been shown to act as a positive regulator in T, B, and NK cell signal transduction. 3BP2 is an important regulator of cytotoxic granule release in NK cells. Mast cells (MCs) similarly degranulate following Ag-dependent aggregation of the FcεRI on the cell surface. Activation of these cells induces the release of preformed inflammatory mediators and the de novo synthesis and secretion of cytokines and chemokines. Thus, MCs participate in both innate and acquired responses. We observed that 3BP2 is expressed in human MCs (huMCs) from diverse origins. Moreover, 3BP2 coimmunoprecipitates with essential MC signaling mediators such as Lyn, Syk, and phospholipase C γ; thus, a role for this adaptor in MC function was postulated. In the present work, we used the short hairpin RNA lentiviral targeting approach to silence 3BP2 expression in huMCs. Our findings point to a requirement for 3BP2 in optimal immediate and late MCs responses such as degranulation and IL-8 or GM-CSF secretion. 3BP2 was determined to be necessary for optimal phosphorylation of Syk, linker for activation of T cells, and phospholipase C γ(1), critical signals for calcium release from intracellular stores. Taken together, our results show that by participating in FcεRI- mediated signal transduction 3BP2 is an important regulator of huMC activation. Thus, 3BP2 could be a potential therapeutic target for IgE-dependent MC-mediated inflammatory disease.     

Hormonal induction of tyrosine aminotransferase and RNA synthesis in the cells of Morris hepatoma strain 7777

The transfer of Morris hepatoma cells induced by the hormone within 10-60 min in to a hormone-free medium is associated with the augmentation of tyrosine aminotransferase synthesis. The kinetics of this process does not differ from that of the hormone-induced enzyme. The return of tyrosine aminotransferase synthesis to the basal level occurs 15-20 hours after the hormone withdrawal from the medium, although the concentration of the intranuclear hormone sharply decreases already after 3 hours. It was demonstrated that the presence in the hepatoma cell nuclei of 20-25% of the initially bound hormone for at least 20 hours after the cell transfer to the hormone-free medium is not sufficient for maintaining a high level of tyrosine aminotransferase gene expression. Using two-dimensional electrophoresis of 3H-labeled hepatoma cell proteins, it was demonstrated that the observed high activity of tyrosine aminotransferase is due to the de novo synthesis of enzyme molecules rather than to the existence of preformed long-living tyrosine aminotransferase molecules inside the cell. Study of [14C]uridine incorporation into non-ribosomal nuclear RNA of hepatoma cells showed a long-term presence of the label in the RNA throughout the chase experiment. It was assumed that the high activity of the enzyme for 10-15 hours after the hormone release from the hepatoma cell nuclei is due to the accumulation in the nuclei of long-living pre-mRNA molecules synthesized after the hormone addition to the cells and during the first hours after the cell transfer to the hormone-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of urokinase A chain with the receptor of human keratinocytes stimulates release of urokinase-like plasminogen activator

On the basis of a fibrinolytic assay with 125I-fibrin, zymography, and immunoprobing with anti-human urokinase antibody, we have observed that the in vitro established NCTC human keratinocyte cell line releases into the culture medium a 54,000-Da plasminogen activator which is indistinguishable from human urokinase. Only the early release following the washing of keratinocyte monolayers is accounted for by secretion of preformed enzyme, while late secretory events require the de novo synthesis of urokinase. The released enzyme can interact by autocriny with its own receptor present on keratinocytes. The addition to the keratinocyte culture medium of the urokinase A chain can stimulate a concentration-dependent urokinase oversecretion, which is not paralleled by oversecretion of plasminogen activator inhibitor-1. Since stimulation of urokinase production can be obtained by an A chain concentration (5 ng/ml) which was previously shown to be efficient in inducing keratinocyte mobilization in an in vitro migration model system, we hypothesize that this mechanism may be important in vivo during the process of wound repair.     

Newly Synthesized and Preformed Acetylcholine Are Released from Torpedo Synaptosomes by Different Pathways

In this study, we investigated the mechanisms underlying the release of preformed and of newly synthesized acetylcholine (ACh) from isolated Torpedo nerve terminals (synaptosomes). This was pursued by examining and comparing the effects of anticytoskeletal and anticalmodulin drugs and of activating the presynaptic muscarinic ACh receptors on the release of preformed endogenous ACh and of newly synthesized radiolabeled ACh. The anticytoskeletal drugs vinblastine, cytochalasin B, and colchicine inhibit the Ca2+-dependent K+-mediated release of newly synthesized radiolabeled ACh, but have no effect on the release of preformed ACh. By contrast, the muscarinic agonist oxotremorine markedly inhibits the release of preformed ACh, but has little effect on the release of newly formed ACh. Treatment of the synaptosomes with the calmodulin antagonist trifluoperazine inhibits the release of both ACh pools concomitantly. These findings show that preformed and newly synthesized ACh are released by different routes and suggest that their secretion is mediated by converging pathways. The significance of these results in view of the previously demonstrated preferential release of newly synthesized ACh is discussed.     

Synthesis and secretion of hemolysin by Escherichia coli.

Hemolytic Escherichia coli cells were found to synthesize and secrete significant amounts of hemolysin into a mineral salt-glucose medium containing hemoglobin. The release of de novo-synthesized hemolysin was stopped in the presence of energy metabolism inhibitors such as 2,4-dinitrophenol, sodium azide, or potassium cyanide, resulting in an accumulation of intracellular hemolysin. A similar effect was observed in the presence of procaine, a neuroactive drug which inhibits the processing of exoproteins. Small amounts of hemolysin were secreted into the medium within approximately 10 min of inhibition of protein synthesis by chloramphenicol. This represented the final release of preformed periplasmic hemolysin en route to secretion through the outer membrane and was not caused by adsorption of external hemolysin to the cell surface. This secretion was not energy dependent but was inhibited above pH 8 and at low temperatures (10 to 20 degrees C). We concluded that two transport processes are involved in hemolysin secretion. De novo-synthesized hemolysin is extruded by an energy-dependent process through the cytoplasmic membrane and probably requires processing. In the periplasmic space a small internal pool of preformed hemolysin is accumulated temporarily before being transported through the outer membrane. Release of hemolysin through the outer membrane does not require energy or de novo protein synthesis.     

Proteinase-activated receptor-1 regulation of macrophage elastase (MMP-12) secretion by serine proteinases

The serine proteinases plasmin and thrombin convert proenzyme matrix metalloproteinases (MMPs) into catalytically active forms. In addition, we demonstrate that plasmin(ogen) and thrombin induce a significant increase in secretion of activated murine macrophage elastase (MMP-12) protein. Active serine protease is responsible for induction, as demonstrated by the absence of MMP-12 induction in plasminogen(Plg)-treated urokinase-type plasminogen activator-deficient macrophages. Since increased MMP-12 protein secretion was not accompanied by an increase in MMP-12 mRNA, we examined post-translational mechanisms. Protein synthesis was not required for early release of MMP-12 but was required for later secretion of activated enzyme. Immunofluorescent microscopy demonstrated basal expression in macrophages that increased following serine proteinase exposure. Inhibition of MMP-12 secretion by hirudin and pertussis toxin demonstrated a role for the thrombin G protein-coupled receptor (protease-activated receptor 1 (PAR-1)). PAR-1-activating peptides were able to induce MMP-12 release. Investigation of signal transduction pathways involved in this response demonstrate the requirement for protein kinase C, but not tyrosine kinase, activity. These data demonstrate that plasmin and thrombin regulate MMP-12 activity through distinct mechanisms: post-translational secretion of preformed MMP-12 protein, induction of protein secretion that is protein kinase C-mediated, and extracellular enzyme activation. Most importantly, we show that serine proteinase MMP-12 regulation in macrophages occurs via the protein kinase C-activating G protein-coupled receptor PAR-1.     

Regulation of the leptin content of obese human adipose tissue

The objective of this study was to determine whether obese human adipose tissue contains preformed stores of leptin and their relationship to secreted leptin. Detergent increased detectable leptin by about twofold, suggesting that leptin is stored in a membrane-bound location. Subcutaneous tissue leptin was approximately 1.6-fold higher than omental, paralleling known differences in leptin secretion and expression. The amount of leptin secreted during a 3-h incubation was similar to that of extractable tissue leptin. Tissue leptin levels were maintained over the incubation. Inhibition of protein synthesis decreased tissue leptin content but did not decrease leptin secretion until after 3 h of incubation. Culture of adipose tissue for 2 days with the combination of insulin and dexamethasone, but not with either hormone alone, increased tissue leptin content about twofold in both depots. Although insulin did not affect tissue leptin content, it potentiated leptin secretion (as a % of tissue stores). These data suggest that adipose tissue leptin storage and secretion per se are regulated. Regulation of the release of preformed leptin may modulate serum leptin levels in obese humans.     

Control of Angiogenesis by Galectins Involves the Release of Platelet-Derived Proangiogenic Factors

Platelets contribute to vessel formation through the release of angiogenesis-modulating factors stored in their α-granules. Galectins, a family of lectins that bind β-galactoside residues, are up-regulated in inflammatory and cancerous tissues, trigger platelet activation and mediate vascularization processes. Here we aimed to elucidate whether the release of platelet-derived proangiogenic molecules could represent an alternative mechanism through which galectins promote neovascularization. We show that different members of the galectin family can selectively regulate the release of angiogenic molecules by human platelets. Whereas Galectin (Gal)-1, -3, and -8 triggered vascular endothelial growth factor (VEGF) release, only Gal-8 induced endostatin secretion. Release of VEGF induced by Gal-8 was partially prevented by COX-1, PKC, p38 and Src kinases inhibitors, whereas Gal-1-induced VEGF secretion was inhibited by PKC and ERK blockade, and Gal-3 triggered VEGF release selectively through a PKC-dependent pathway. Regarding endostatin, Gal-8 failed to stimulate its release in the presence of PKC, Src and ERK inhibitors, whereas aspirin or p38 inhibitor had no effect on endostatin release. Despite VEGF or endostatin secretion, platelet releasates generated by stimulation with each galectin stimulated angiogenic responses in vitro including endothelial cell proliferation and tubulogenesis. The platelet angiogenic activity was independent of VEGF and was attributed to the concerted action of other proangiogenic molecules distinctly released by each galectin. Thus, secretion of platelet-derived angiogenic molecules may represent an alternative mechanism by which galectins promote angiogenic responses and its selective blockade may lead to the development of therapeutic strategies for angiogenesis-related diseases.     

Effect of dexamethasone, indomethacin, and lipopolysaccharide on the secretion of interstitial collagenase and type V collagenase by cultured macrophages

The rabbit alveolar macrophage secretes at least two collagenolytic metalloproteinases in vitro including an interstitial collagenase and a type V collagenase. Using assays previously shown to discriminate between these two activities, the secretion of these two enzyme activities was investigated. Both enzyme activities accumulated in culture over 11 days and the release of both were similarly inhibited by cycloheximide. Collagenolytic activity was negligible in cell lysates. The interstitial collagenase was found in a latent form but the type V collagenase activity was active in the culture medium. When cultured in the presence of dexamethasone, the secretion of both the enzymes were identically inhibited in a dose-dependent manner. Indomethacin was an effective inhibitor of secretion of both collagenases at a concentration of 10(-5) M but not at lower concentrations. Finally, bacterial lipopolysaccharide stimulated the secretion of both type V and interstitial collagenase by these cells. These studies indicate that, like the interstitial collagenase, the type V collagenase is released from the cell as synthesized and is not stored intracellularly. Protein synthesis is necessary for the release of both these collagenases. Furthermore, the release of type V collagenase responded to dexamethasone, indomethacin, and lipopolysaccharide in a manner identical to the secretion of the interstitial collagenase suggesting that synthesis and secretion of these two enzymes are regulated in a similar manner.     

Amyloid precursor protein and amyloid beta peptide in human platelets. Role of cyclooxygenase and protein kinase C

The main component of Alzheimer's disease (AD) senile plaques is amyloid-beta peptide (Abeta), a proteolytic fragment of the amyloid precursor protein (APP). Platelets contain both APP and Abeta and may contribute to the perivascular amyloid deposition seen in AD. However, no data are available concerning the biochemical mechanism(s) involved in their formation and release by these cells. We found that human platelets released APP and Abeta following activation with collagen or arachidonic acid. Inhibition of platelet cyclooxygenase (COX) reduced APP but not Abeta release following those stimuli. In contrast, activation of platelets by thrombin and calcium ionophore caused release of both APP and Abeta in a COX-independent fashion. Ex vivo studies showed that, despite suppression of COX activity, administration of aspirin did not modify Abeta or APP levels in serum or plasma, suggesting that this enzyme plays only a minor role in vivo. We examined the regulation of APP cleavage and release from activated platelets and found that cleavage requires protein kinase C (PKC) activity and is regulated by the intracellular second messengers phosphatidylinositol 2-phosphate and Ca(2+). Our data provide the first evidence that in human platelets COX is a minor component of APP secretion whereas PKC plays a major role in the secretory cleavage of APP. By contrast, Abeta release may represent secretion of preformed peptide and is totally independent of both COX and PKC activity.     

Intracellular or extracellular calcium can be used to trigger C5a-stimulated enzyme secretion from rabbit neutrophils

The ability of C5a to stimulate lysosomal enzyme release and 45Ca2+ efflux from rabbit neutrophils was studied. C5a stimulated beta-glucuronidase release from cytochalasin B-treated neutrophils either in the presence or absence of extracellular calcium. Depletion of cell calcium by pretreatment with the calcium ionophore A23187 blocked both the ability of C5a to elicit enzyme release in the absence of extracellular calcium and its ability to stimulate 45Ca2+ efflux. Both actions were dose-dependent over the same concentration range (10(-8)-10(-6) M ionophore A23187). In contrast, ionophore pretreatment had no effect on C5a-stimulated enzyme release in the presence of extracellular calcium. These results suggest that (a) release of cell calcium is required for enzyme secretion in the absence of extracellular calcium, and (b) C5a can trigger near-maximal enzyme release by using calcium from either of two sources: the extracellular space or an intracellular site.     

Secretion of metalloendopeptidase 24.15 (EC 3.4.24.15).

The metalloendopeptidase EP24.15 (EC3.4.24.15) is a neuropeptide-metabolizing enzyme present in neural and endocrine tissues, presumably functioning extracellularly. Because the majority of the EP24.15 activity is identified in the soluble fraction of cellular homogenates, suggesting that the enzyme is primarily an intracellular protein, we addressed the issue of how EP24.15 arrives in the extracellular environment. We utilized a model system of neuroendocrine secretion, the AtT20 cell. According to both enzymatic activity and immunologic assays, EP24.15 was synthesized in and released from AtT20 cells. Under basal conditions and after stimulation by corticotropin-releasing hormone or the calcium ionophore A23187, EP24.15 activity accumulated in the culture medium. This secretion was not attributable to cell damage, as judged by the absence of release of cytosolic enzyme markers and the ability to exclude trypan blue dye. Pulse-chase analysis and subcellular fractionation of AtT20 cell extracts suggested that the mechanism of EP24.15 secretion is not solely via classical secretory pathways. Additionally, drugs which disrupt the classical secretory pathway, such as Brefeldin A and nocodazole, blocked A23187-stimulated EP24.15 release yet had no effect on basal EP24.15 release, suggesting differences in the basal and stimulated pathways of secretion for EP24.15. In summary, EP24.15 appears to be secreted from AtT20 pituitary cells into the extracellular milieu, where the enzyme can participate in the physiologic metabolism of neuropeptides.     

A continuous flow method for the study of lipoprotein lipase secretion in adipose cells

The secretion of lipoprotein lipase has been examined in Ob17 adipose cells. No spontaneous secretion is detected. The activity of the heparin-releasable enzyme shows a first-order process of inactivation. This constant rate of inactivation, coupled with a decreased rate of secretion, prevents any significant determination of enzyme secretion in heparin-containing media. Thus, a perifusion system, with which the rate of enzyme inactivation is minimal and systematic, has been devised and used. The data show that the secretion of a pool of pre-existing lipoprotein lipase molecules is followed by the secretion of newly synthesized enzyme molecules. The results are discussed with respect to the significance of the determinations of the heparin-releasable enzyme in most studies as well as with respect to the intracellular localization of lipoprotein lipase in Ob17 cells.     

Cell-density-dependent release of hepatic lipase from cultured rat hepatocytes

Cultured rat hepatocytes release the enzyme hepatic lipase. In this study we investigated the effect of cell density on this metabolic function under a variety of experimental conditions. The release of hepatic lipase from cultured rat hepatocytes exhibits a cell-density dependence, the secretion per mg cell protein being increased with increasing cell density. When cell density dependence was taken into consideration no significant effect of insulin on the release of hepatic lipase from cultured hepatocytes was observed, whereas glucagon suppressed the release. Glucose stimulating the release of the enzyme, especially in cultures with high cell density.     

Induction of lysosomal enzyme secretion by alveolar macrophages in response to the purified complement fragments C5a and C5a des-arg

Purified C5a and its "inactive" form, C5a des-arg, were shown to induce secretion of acid hydrolases from rabbit alveolar macrophages (AM) in a concentration-dependent manner. Secretion increased with time to 5 times above controls by 72 hr. Concentrations of these enzymes in the cell lysates did not decrease during the incubation, suggesting that synthesis of new enzyme was occurring. The lysosomal enzyme secretion was accompanied by increased pinocytosis and release of proteolytic enzymes from the macrophages. At no time was significant lactic dehydrogenase liberated, indicating that secretion was selective and not due to cell death. Data presented also suggest that C5a des-arg induced secretion from the macrophages of a chemotactic factor for neutrophils. It was concluded that C5a and C5a des-arg may play a role in lung injury by interactions with AM, inducing the secretion of acid hydrolases and proteolytic enzymes that can cause tissue damage, and by regulating the influx of other inflammatory cells into the interstitium and air spaces.     

Release of chemical transmitters from cell bodies and dendrites of nerve cells

Papers in this issue concern extrasynaptic transmission, namely release of signalling molecules by exocytosis or diffusion from neuronal cell bodies, dendrites, axons and glia. Problems discussed concern the molecules, their secretion and importance for normal function and disease. Molecules secreted extrasynaptically include transmitters, peptides, hormones and nitric oxide. For extrasynaptic secretion, trains of action potentials are required, and the time course of release is slower than at synapses. Questions arise concerning the mechanism of extrasynaptic secretion: how does it differ from the release observed at synaptic terminals and gland cells? What kinds of vesicles take part? Is release accomplished through calcium entry, SNAP and SNARE proteins? A clear difference is in the role of molecules released synaptically and extrasynaptically. After extrasynaptic release, molecules reach distant as well as nearby cells, and thereby produce long-lasting changes over large volumes of brain. Such changes can affect circuits for motor performance and mood states. An example with clinical relevance is dyskinesia of patients treated with l-DOPA for Parkinson''s disease. Extrasynaptically released transmitters also evoke responses in glial cells, which in turn release molecules that cause local vasodilatation and enhanced circulation in regions of the brain that are active.